Process for chenodeoxycholic acid and intermediates therefore

ABSTRACT

A multi-step synthesis of chenodeoxycholic acid from 3-keto-bisnorcholenol, a compound readily obtained from the abundant plant sterol β-sitosterol, is described. A key step in the synthesis is the stereoselective microbial introduction of the 7-alpha hydroxy group into 3-keto-bisnorcholenol.

BACKGROUND OF THE INVENTION

Chenodeoxycholic acid has exhibited valuable therapeutic activity,particularly as an agent for dissolution of cholesterol gallstones. Thiscompound is currently obtained from cholic acid. An exemplary synthesisstarting from cholic acid is described in U.S. Pat. No. 3,836,550.However, this process is of limited importance due to the circumstanceswhich exist regarding the natural sources of cholic acid which isextracted from cow bile or with some additional processing from chickenbile. Calculations based on estimated demand for chenodeoxycholic acidassuming only one-third the potential patients utilize this drugindicate that such natural sources even if utilized to maximum potentialcould provide only a minor portion of such demand. Thus, an efficientsynthesis from highly abundant starting materials is an important factorin determining whether chenodeoxycholic acid achieves its potential rolein medicine.

Hydroxylation at the 7-position of compounds in the androstane andpregnane series in well known in the art. An early report by Kramli andHorvath, Nature, 4120, 619 (1948) indicated the 7-hydroxylation ofcholesterol by incubation with Proactinomyces roseus was carried out butthe actual configuration of the product was not determined. Welldocumented reports of 7-alpha-hydroxylation on the following substratesappear in the art:

deoxycorticosterone--

Meystre et al., Helv. Chim. Acta 38, 381 (1955)

progesterone and related compounds--

U.S. Pat. No. 2,753,290

U.S. Pat. No. 2,836,608 McAleer et al., J. Org. Chem. 23, 958 (1958)

testosterones--

U.S. Pat. No. 2,801,251

U.S. Pat. No. 2,960,436 Irmscher et al., Chemische Berichten 97, 3363(1964)

A-nor steroids--

U.S. Pat. No. 3,005,018 Laskin and Weisenborn, Bact. Proc. 26, A26(1962)

17-alkyl androstanes and pregnenes--

Singh et al., Can. J. of Microbiol. 13, 1271 (1967)

androstenedione--

Abdul-Hajj. Lloydia 33(2), 278 (1970)

estradiols--

Chem. Abstracts 86, 73006s (1977)

botryodiplodia theobromae is known to be capable of11-alpha-hydroxylating steroids (U.S. Pat. No. 3,047,470). Lasiodiplodiatheobromae and Botryodiplodia theobromae are effective agents for thereduction of pyridine and of pyrimidine compounds. See for example Howeand Moore, J. Med. Chem. 14 (4), 287 (1971); British Pat. No. 1,183,850;and Howe et al., J. Med. Chem. 15, 1040 (1972). Lasiodiplodia theobromaehas also been reported to oxidize mycophenolic acid. Jones et al., J.Chem. Soc. (C), 1725 (1970).

DESCRIPTION OF THE INVENTION

The present invention relates to an efficient synthesis ofchenodeoxycholic acid having the structure below ##STR1## starting from3-keto-bisnorcholenol (also named22-hydroxy-23,24-bisnorchol-4-en-3-one) a compound of the formula:##STR2## which is readily obtained by the microbiological degradation ofthe commercially available β-sitosterol by procedures well known to theart.

In the initial process step of the present invention3-keto-bisnorcholenol is microbiologically hydroxylated in the7-position to produce 7-alpha-hydroxy-3-ketobisnorcholenol(7α,22-dihydroxy-23,24-bisnorchol-4-en-3-one) of the formula: ##STR3##

Of the 152 cultures examined for 7-alpha hydroxylation, such culturesrepresenting 92 species in 41 genera and including many reported capableof 7-alpha hydroxylation of the various substrate compounds enumeratedabove, it has now been unexpectedly found that only 9 very closelyrelated cultures, Botryodiplodia theobromae IFO 6469, ATCC 28570, DSM62-678, DSM 62-679; Botryosphaeria ribis ATCC 22802, B. berengerianaATCC 12557, B. rhodina CBS 374.54, CBS 287.47 and CBS 306.58, arecapable of carrying out the desired 7-alpha hydroxylation on this sterolsubstrate.

The microorganism may be used in the form of the culture broth, themycelia or an enzyme extract thereof. The culture broth may be preparedby inoculating the organism into a suitable medium. The culture mediumcan contain carbon sources, nitrogen sources, inorganic salts and othernutrients suitable for the growth of the microorganisms. The carbonsources are, for example, glucose, sucrose, dextrin, mannose, starch,lactose, glycerol and the like, the nitrogen sources are e.g.nitrogen-containing organic substances, such as peptone, meat extract,yeast extract, corn steep liquor, casein and the like, ornitrogen-containing inorganic compounds, such as nitrates, inorganicammonium salts and the like, and the inorganic salts such as phosphatesor minerals such as sodium, potassium, magnesium, manganese, iron,copper and the like.

As the cultivation method employed in this process step of the inventionthere can be utilized submerged culture, shaking culture, stationaryculture and the like. However, since aerobic conditions are required fororganisms used in this invention, it is preferable to cultivate underconditions which promote aeration.

In addition it is possible to employ in the practice of the presentinvention a mycelium isolated from the culture broth of themicroorganism, or a crude enzyme extracted from the culture broth or themycelium by a known method per se can be brought into contact with thesubstrate under suitable conditions. In the case when such a processembodiment is adopted the 7α-hydroxylation can be conveniently performedin an aqueous solution such as a buffer solution, a physiological saltsolution or a fresh medium, or in water.

The substrate compound may be added in the form of an unpalpable powderor in the form of a solution dissolved in a hydrophilic solvent such asacetone, dimethylsulfoxide, methanol, ethanol, ethylene glycol,propylene glycol, dioxane and the like. Alternatively, a surfactant or adispersing agent may be added to a water suspension of the substrate.Furthermore, the substrate can be prepared as a finely dividedsuspension by treatment with ultrasonic waves.

The fermentation procedure employs conventional techniques. Thus, thedesired microorganism may be grown in Edamin broth (same as fermentationmedium; see below) for a period of from 18 to 72 hours at a temperaturein the range of about 15° to 35° C. Fermentation is initiated byinoculating a conventional fermentation medium with from 1 to 10 wt % ofthe vegetative growth. The fermentation conditions can be the same aswas utilized to grow the inoculum. After an incubation period of from 18to 96 hours, the substrate bisnorcholenol is added either as a solutionin, preferably absolute ethanol or as a sonically prepared solution in0.1% Tween 80 (polyoxyethylene sorbitan monooleate). The fermentationmay be carried out for up to 120 hours after addition of the substrate.A suitable fermentation medium is obtained by mixing the following ormultiple thereof. Edamin (Sheffield Chemical Co.), an enzymatic digestof lactalbumin 20 grams, cornsteep liquor 3 grams, dextrose 50 grams anddistilled water to a final volume of 1 liter. The pH of the medium isadjusted to about 4 to 7, preferably about 5.0 prior to sterilizatione.g. by autoclaving.

Isolation of the desired 7-alpha-hydroxy-3-keto-bisnorchlorenol productfrom the fermentation medium is readily accomplished using procedureswell known in the art. Thus, the harvested whole culture broth can beextracted with a non-miscible organic solvent such as preferably ethylacetate. The solvent soluble fractions may then be purified using gelchromatography such as for example with silica gel G-60, followed bycrystallization.

It has futher been found that a number of procedures can be employed tooptimize the yield of desired product from the fermentation. Thus, forexample, addition of a chelating agent such as 2,2'-dipyridyl in a finalconcentration ranging from 0.5×10⁻⁴ M to 0.75×10⁻³ M, addition with thesubstrate of either glucose or sucrose in a final concentration of about5%, lowering the temperature of the incubation fermentation to about 24°C. after adding the substrate and by using a suspension of substrate ata concentration of 5% in 0.1% Tween 80. An even greater increase inyield is obtainable by the addition of adsorbants to the fermentationmedium. For example yield improvement is obtained when polymeric resinabsorbents such as Amberlite XAD7 (Rohm & Haas Co.), a polymer of themethyl ester of acrylic acid is added at a concentration 0.3-0.6 wt % toa fermentation medium where the substrate is present in a concentrationof up to about 1 g/liter. Best yield improvement was obtained at aboutthe 0.6 wt % concentration level for the adsorbent.

The 7-alpha-hydroxy-3-keto-bisnorcholenol produced by the abovedescribed fermentation procedure is then catalytically hydrogenated soas to produce (5β)7α,22-dihydroxy-23,24-bisnorhcholan-3-one of theformula: ##STR4##

A suitable catalyst for this hydrogenation is palladium preferably on asolid support, preferably 5% palladium on charcoal. The reaction iscarried out in a nonaqueous polar solvent such as dimethylformamide andat a temperature in the range of from about 0° to 50° C., preferably atabout room temperature an at ambient pressure. Isolation of the productcan be carried out in the same manner as from the above fermentation,i.e. gel chromatography such as with silica gel 60 and crystallization.

The saturated product of formula IV is then reacted with a p-tolyl ormethyl sulfonyl halide at a temperature in the range of from 78° to 0°C., preferably at about -10° C. The reaction is conveniently carried outin a nitrogeneous organic solvent such as pyridine. A preferred reagentfor this reaction is p-toluenesulfonyl chloride. The resulting productof this reaction, which can be isolated in the same manner as previouslydescribed for compounds above, has the formula ##STR5## where X ismethyl or p-tolyl, preferably p-tolyl.

A preferred embodiment of a compound of formula V is thus(5β)-7α-hydroxy-22-([4-methylphenyl]sulfonyl-oxy)-23,24-bisnorcholan-3-one.

In the next step of the process of the present invention a compound offormula V is reacted with sodium di-C₁₋₃ -alkyl malonate, preferablydimethylmalonate in a polar non-aqueous solvent such asdimethylformamide at a temperature in the range of from 0° to 100° C.,preferably at about 50° C. in an inert atmosphere with exclusion ofmoisture. The sodium di-C₁₋₃ -alkyl malonate can be prepared in situ byadding the malonate to a solution of sodium hydride in dimethylformamideand stirring at 40°-50° C. Isolation of the end product is carried outin analogy to the procedures described previously. The resulting producthas the formula ##STR6## where Y is C₁₋₃ -alkyl, preferably methyl. Apreferred embodiment of a compound of formula VI is thus(5β)-24-norcholan-7α-ol-3-one-23,23-dicarboxylic acid dimethyl ester.

The compound of formula VI is then reduced to the corresponding3-hydroxy compound of the formula ##STR7## where Y is C₁₋₃ -alkyl,preferably methyl. A preferred embodiment of a compound of formula VIIis (5β)-24-norcholane-3α,7α-diol-23,23-dicarboxylic acid dimethyl ester.

The reduction procedure is accomplished by using a conventional chemicalreducing agent such as sodium borohydride in an aqueous C₁₋₃ alkanolsolvent such as 95% ethanol at a temperature in the range 0° to 50° C.,preferably at room temperature under an inert atmosphere. The reactionproduct can be isolated from the reaction mixture by acidifying andextracting with a halocarbon solvent such as dichloroethane. Removal ofthe solvent provides the product in crude form which can be used withoutfurther purification in succeeding steps.

The diol of formula VII is then saponified by refluxing in the presenceof strong base, i.e. barium hydroxide so as to provide, after work-upand acidification a dicarboxylic acid of the formula ##STR8## which is(5β)-24-norcholane-3α,7α-diol-23,23-dicarboxylic acid.

In the final step of this embodiment of process of the invention thedicarboxylic acid of formula VIII is thermally decarboxylated by heatingthe formula VIII compound at a temperature of about 190°-205° C. underan inert atmosphere so as to produce the desired end productchenodeoxycholic acid of formula I above.

In an alternate process embodiment of the present invention a compoundof formula V can be treated with a chemical reducing agent such as alithium aluminum alkoxide hydride, preferably lithium aluminumtri-t-butoxyhydride, at a temperature in the range of from about -78° C.to room temperature, preferably at about -10° C. in an inert organicsolvent such as a cyclic ether, preferably tetrahydrofuran and under aninert atmosphere so as to produce a diol of the formula ##STR9## where Xis as above.

A preferred embodiment of a compound of formula IX is2,2-([4-methylphenylsulfonyl]oxy)-23,24-bisnorcholane-3α,7α-diol.

In the next process step the compound of formula IX is reacted with agreater than twofold molar excess of an acylating agent conventionallyemployed as a hydroxy protecting group in steroid chemistry so as toprepare the corresponding diacyl compound. Suitable acylating agentsinclude the C₂₋₆ lower alkanoic acid anhydrides, preferably aceticanhydride. The acylation is readily carried out in a suitable organicsolvent such as pyridine in the presence of an amine base such as4-dimethylaminopyridine. The resulting diacyl product has the formula:##STR10## where X is as above and R is acyl.

A preferred embodiment of a compound of formula X is22-([(4-methylphenyl)sulfonyl]oxy)-23,24-bisnorcholane-3α,7α-diol3,7-diacetate.

The diacyl compound of formula X is then reacted with a sodium di-C₁₋₃-alkyl malonate, preferably diethylmalonate in direct analogy to thepreviously described conversion of compound V to compound VI above so asto produce a compound of the following formula: ##STR11## where R and Yare as above.

A preferred embodiment of a compound of formula XI is3α,7α-(diacetoxy)-24-norcholane-23,23-dicarboxylic acid diethyl ester.

Conversion of a compound of formula XI to a dicarboxylic acid of formulaVIII described above is readily accomplished by saponification with astrong base such as an alkali metal hydroxide solution, preferablypotassium hydroxide at elevated temperature, preferably at reflux. Thereaction can be carried out in the presence of one or more loweralkanols as solvent such as, for example methanol, isopropanol ormixtures thereof.

The processes and intermediates of the present invention are furtherillustrated by reference to the following Examples.

EXAMPLE 1(5β)-7α,22-Dihydroxy-23,24-bisnorchol-4-en-3-one(7-alpha-OH-3-KC)Fermentation procedures

Cultures were maintained on the following media: bacteria, glucosenutrient agar; fungi; Sabouraud Dextrose agar (SD) (Difco);actinomycetes, starch-casein agar. Vegetative inoculum was prepared ineither Sabouraud Dextrose broth (Difco) or in the medium used in thefermentation stage. The latter was composed of: Edamin (SheffieldChemical Co.), 20 g; cornsteep liquor (Corn Products Co.), 3 g.;dextrose, 50 g.; and distilled water to a final volume of 1 liter. ThepH was adjusted to 5.0 with HCl prior to sterilization by autoclaving.The fermentations were carried out either in 250 or 500 ml Erlenmeyerflasks containing 50 and 100 ml of medium respectively. The flasks wereinoculated with vegetative growth (5%) from cultures grown in theinoculum medium for 72 hours at 28° C. on a 250 RPM rotary shaker (5 cmeccentricity). Unless otherwise indicated, the same conditions apply forthe fermentation stage. The 3-keto-bisnorcholenol (3-KC) was added after48 hour incubation either as a solution in 3A ethanol, or as a 5%suspension in 0.1% solution of Tween 80 (Atlas Chemical Industries)prepared by sonic treatment (Bronson Sonifier Cell Disruptor 200).Incubation was continued for up to 120 hours after addition of thesubstrate.

Seventy-five species of fungi representing thirty-two genera, sixspecies of actinomycetes, representing five genera, and eight species ofgram-negative bacteria belonging to three genera, and three species ofGram positive bacteria belonging to one genus (152 cultures in total),were tested for their ability to convert 3-KC to 7-alpha-OH-3-KC. As aresult of this screening, only nine cultures were found capable of thedesired transformation. Other members of these same genera, and, in somecases, other strains of the same species, appeared to be incapable ofconverting the substrate to the desired product (Table 1).

    ______________________________________                                                         Source                                                       Culture          Code       7-α-OH-3-KC                                 ______________________________________                                        Botryodiplodia theobromae                                                                      IFO 6469   +                                                 Botryodiplodia theobromae                                                                      DSM 62-678 +                                                 Botryodiplodia theobromae                                                                      DSM 62-679 +                                                 Lasiodiplodia theobromae                                                      (Botryodiplodia theobromae)                                                                    ATCC 28570 +                                                 Lasiodiplodia theobromae                                                      (Diplodia natalensis)                                                                          ATCC 9055  0                                                 Lasiodiplodia theobromae                                                      Diplodia theobromae)                                                                           ATCC 10936 0                                                 Lasiodiplodia theobromae                                                      (Botryodiplodia theobromae)                                                                    ATCC 16931 0                                                 Lasiodiplodia theobromae                                                      (Botryodiplodia theobromae)                                                                    ATCC 26123 0                                                 Diplodia natalensis                                                                            ATCC 9055  0                                                 Diplodia zeae    QM 6983    0                                                 Botryodiplodia malorum                                                                         CBS 134.50 0                                                 Botryosphaeria ribis                                                                           ATCC 22802 +                                                 Botryosphaeria berengeriana                                                                    ATCC 12557 +                                                 Botryosphaeria rhodina                                                                         CBS 374.54 +                                                 Botryosphaeria rhodina                                                                         CBS 287.47 +                                                 Botryosphaeria rhodina                                                                         CBS 306.58 +                                                 Botryosphaeria corticis                                                                        ATCC 22927 0                                                 ______________________________________                                    

Shake flask experiments on Botryodiplodia theobromae IFO 6469 indicatedthat product yields were increased by addition of 2,2'-dipyridyl, achelating agent, to final concentrations ranging from 0.5×10⁻⁴ M to0.75×10³ M.

Also, it was noted that at the time of substrate addition, 48 h., theculture was depleted of the glucose originally present. Addition ofeither glucose or sucrose (final concentration 5%) at this time appearedto slow degradation of 3-KC and 7-alpha-OH-3-KC. Other smallimprovements in yield were also achieved by lowering the temperature to24° C. at 48 h., and by using a 5% suspension of substrate prepared bysonic treatment in 0.1% Tween 80. By combining all of these improvedconditions, a 25% conversion of 3-KC to 7-alpha-hydroxy-3-KC wasachieved with 46% of unreacted 3-KC still present (analyses by highpressure liquid chromatography (HPLC)).

The most striking improvement in yield was found as a result of additionof absorbants to the fermentation. When Amberlite XAD-7 was added at0.3-0.6% to fermentations of Botryodiplodia theobromae IFO 6469 strain,a substantial increase in the yield of 7-alpha-OH-3-KC resulted. Thisincrease of product formation was evident at 3-KC concentrations of upto 1 g/liter; at higher substrate concentrations XAD-7 was withouteffect. Similarly, no further stimulation of yields were obtained withXAD-7 in amounts above 0.6%. Before use, the XAD-7 resin was refluxed inacetone for 21/2 hours, rinsed repeatedly with distilled water until alltraces of acetone and color were gone, and dried at 40° C.

The 7-alpha-hydroxylation of 3-KC was also carried out with the saidLasiodiplodia theobromae culture with minor modifications of the basicfermentation procedures. HPLC analyses of an extract of a fermentationcarried out with this organism indicated a 25% yield with 22% ofremaining substate.

Identification of the transformation product of 3-KC was performed onmaterial isolated from both cultures as described below.

Isolation of 7-alpha-OH-3-KC

Two liters of harvested whole culture broth from a fermentation whichhad been charged with 1 gram of 3-KC, was extracted twice, each timewith 1 liter of ethyl acetate. The extracts were combined, concentratedto 0.5 liter and filtered through glass wool. The filtrate wasevaporated to dryness and the residue redissolved in 150 ml of hot ethylacetate. After cooling to room temperature an insoluble fraction wasagain removed by filtration. The filtrate was concentrated byevaporation to 35 ml and applied to a 29 mm dia. column containing 200 gof silica gel G-60. The column was developed with ethyl acetate and the7-alpha-OH-3-KC rich fractions combined, concentrated andrechromatographed on silica gel G-60 developed with methylene chloride,ethyl acetate, hexane (1:1:1). The 7-alpha-OH-3-KC rich fractions wereagain combined, the solvent removed by evaporation and the productobtained by crystallization from a small volume of ethyl acetate (220 mgfrom the Botryodiplodia culture; 185 mg from the Lasiodiplodia culture).Identity as 7-alpha-OH-3-KC was established by comparison with anauthentic synthetic sample of 7-alpha-OH-3-KC: m.p. 199°-200° C.(authentic sample 197.5°-200° C.); mixture m.p. (undepressed); NMR, massspectrum and specific rotation.

Analyses

Unless otherwise indicated, quantitative analyses for 3-KC and7-alpha-OH-3-KC were by thin-layer chromatography (TLC). These werecarried out on ethyl acetate extracts of fermentation samples. Theextracts were evaporated to dryness at 40° C. and redissolved in avolume of 3A ethanol ten times smaller than the original sample volume.Chromatography was on silica gel F₂₅₄ TLC plates (E. Merck, Darmstadt,Germany) developed with ethyl acetate. The developed plates wereair-dried and the spots visualized under short wavelength UV light (254nm). The R_(f) values obtained in this TLC system were 0.75 and 0.36 forsubstrate and product respectively. Minor amounts of products havingR_(f) values lower than 0.36 can be seen in the chromatogram, and theirseparation from the desired product can be improved by repeateddevelopment with the same solvent system.

Quantitative assays of the materials in the spots were made by carefullyscraping the area of the spot from the plate into a test tube, andeluting overnight with 5 ml of 3A ethanol. The absorbance of the eluatewas measured at 240 nm in a Gilford 250 spectrophotometer, and comparedwith values from a standard curve obtained by chromatographing knownamounts of authentic 3-KC and 7-alpha-OH-3-KC. TLC results were alsoquantitated by measuring fluorescence quenching of the spots (Zeiss TLCSpectrophotometer, PMQII).

High pressure liquid chromatographic HPLC) analyses were carried out ona silica gel (SR-I-10) column developed with 20% dioxane in methylenechloride. The column was monitored with a 254 nm detector.

EXAMPLE 2 (5β)-7α,22-Dihydroxy-23,24-bisnorcholan-3-one

To 1.92 g (0.0055 mol) of 7α,22-dihydroxy-23,24-bisnorchol-4-en-3-onedissolved in 25 ml of freshly distilled, dry dimethylformamide was added0.19 g of 5% palladium on carbon. The suspension was stirred under ahydrogen atmosphere at room temperature for 5.5 hr. Hydrogen uptakeceased at 108 ml (theory 124 ml). The catalyst was removed by filtrationand was washed with 200 ml of ethyl acetate. The filtrate was pouredinto 11. of water and was extracted with 5×250 ml of ethyl acetate(saturated brine was added to aid break-up of emulsions). The combinedethyl acetate extracts were washed with 5×250 ml of water followed by2×250 ml of saturated sodium chloride. The ethyl acetate was dried oversodium sulfate and evaporated in vacuo to give 2.13 g of crude(5β)-7α,22-dihydroxy-23,24-bisnorcholan-3-one. The total product waschromatographed on 150 g of silica gel 60 and eluted with ethyl acetate.The product fraction weighing 1.7 g was rechromatographed on 300 g ofsilica gel 60 and eluted with a solvent mixture of ethylacetate/methylene chloride (2:1) giving 1.29 g (67%) of(5β)-7α,22-dihydroxy-23,24-bisnorcholan-3-one. The analytical sample wascrystallized from ethyl acetate, mp 132°-133° C.

[α]²⁵ D=+15.4 (c 1.01, CHCl₃).

Calc for C₂₂ H₃₆ O₃ : C, 75.82; H, 10.41. Found: C, 76.10; H, 10.69

EXAMPLE 3(5β)-7α-Hydroxy-22-([(4-methylphenyl)sulfonyl]-oxy)-23,24-bisnorcholan-3-one

To 1.0 g (0.00286 mol) of (5β)-7α,22-dihydroxy-23,24-bisnorcholan-3-onedissolved in 20 ml of absolute pyridine cooled to -10° C. was added 2.18g (0.0114 mol) of p-toluenesulfonyl chloride. The solution was stirredfor 1 hr at -10° C. and then placed in a refrigerator overnight. Thereaction was then poured into 400 ml of a 0.25 N sodium bicarbonatesolution and extracted with 4×100 ml of ethyl acetate. The combinedethyl acetate extracts were washed successively with 3×100 ml of 1 Nsodium bicarbonate, 3×100 ml of water, 3×100 ml of 1 N hydrochloricacid, and finally with water until neutral. After drying with sodiumsulfate, the ethyl acetate was evaporated to give 1.54 g of crudeproduct which was purified by column chromatography on 150 g of silicagel 60. Elution with benzene/ethyl acetate (4:1) gave 1.15 g (80% yield)of(5β)-7α-hydroxy-22-([(4-methylphenyl)sulfonyl]-oxy)-23,24-bisnorcholan-3-one.The analytical sample was recrystallized from toluene/heptane, mp157°-160° C.,

[α]²⁵ D+12.7 (c 0.994, CHCl₃).

Anal. Calcd: C, 69.29; H, 8.42. Found: C, 69.55; H, 8.47.

EXAMPLE 4 (5β)-24-Norcholan-7α-ol-3-one-23,23-dicarboxylic acid dimethylester

To 3 ml of dry dimethylformamide at room temperature under an argonatmosphere was added 30 mg (0.0007 mol) of 57% sodium hydride followedby 0.066 g (0.0005 mol) of dimethyl malonate dissolved in 1 ml ofdimethylformamide. After stirring for 1.5 hr at 40°-50° C., 0.251 g(0.0005 mol) of(5β)-7α-hydroxy-22-([(4-methylphenyl)sulfonyl]-oxy)-23,24-bisnorcholan-3-onewas added. The reaction was stirred for 1 hr at room temperature, warmedto 50° C. in an oil bath for 18 hr, and then poured into 25 ml of water.Extraction with 4×10 ml of ethyl acetate followed by washes with waterand saturated brine gave, after drying over sodium sulfate andevaporation, 0.20 g of crude product. After chromatography on 20 g ofsilica gel 60 and elution with a mixture of dichloromethane/ethylacetate (2:1), 0.041 g (17% yield) of(5β)-24-norcholan-7α-ol-3-one-23,23-dicarboxylic acid dimethyl ester wasobtained.

EXAMPLE 5 (5β)-24-Norcholane-3α,7α-diol-23,23-dicarboxylic acid dimethylester

To a solution of 0.195 g (0.00042 mol) of(5β)-24-norcholan-3-one-23,23-dicarboxylic acid dimethyl ester in 12 mlof 95% ethanol at room temperature under an argon atmosphere was added0.025 g (0.00063 mol) of sodium borohydride. The reaction mixture wasallowed to stir for 2 hrs, then poured into 50 ml of water containing 4ml of 1 N hydrochloric acid. The aqueous solution was extracted withdichloromethane and the combined extracts dried over sodium sulfate andevaporated to give 0.195 g of the above-captioned crude diol.

EXAMPLE 6 (5β)-24-Norcholane-3α,7α-diol-23,23-dicarboxylic acid

To a solution of 0.195 g of crude(5β)-24-norcholane-3α,7α-diol-23,23-dicarboxylic acid dimethyl ester in5 ml of ethanol was added 2 ml of water followed by 0.80 g of bariumhydroxide. The solution was heated at reflux for 3 hrs, cooled to roomtemperature, and then acidified by the addition of 1 N hydrochloricacid. After extraction with dichloromethane and drying, the solvent wasevaporated to give 0.135 g of(5β)-24-norcholane-3α,7α-diol-23,23-dicarboxylic acid.

EXAMPLE 7 Chenodeoxycholic Acid

In a round-bottom flask under an argon atmosphere, 0.135 g (0.00031 mol)of (5β)-24-norcholane-3α,7α-diol-23,23-dicarboxylic acid was heated for10 min at 190°-205° C. Gas evolution was observed. After cooling, theresidue was chromatographed on 10 g of silica gel 60 and eluted with 10%ethanol in ethyl acetate to give 0.045 g of chenodeoxycholic acid as aglass. Tlc and nmr were identical with an authentic sample.

EXAMPLE 822-([(4-methylphenyl)sulfonyl]oxy)-23,24-bisnorcholane-3α,7α-diol

To 0.350 g (0.00069 mole) of(5β)-7α-hydroxy-22-([(4-methylphenyl)sulfonyl]-oxy)-23,24-bisnorcholan-3-onein 10 ml of dry tetrahydrofuran cooled to -10° C. under an argonatmosphere is added dropwise 0.391 g (0.00138 mole) of lithium aluminumtri-t-butoxyhydride dissolved in 5 ml of dry tetrahydrofuran. After 11/2hr, the reaction was quenched by the addition of 2 ml of 1 Nhydrochloric acid. The tetrahydrofuran was removed in vacuo. The residuewas taken up in 50 ml of water and extracted with 3×40 ml of ethylacetate. The combined ethyl acetate extracts were washed with wateruntil neutral and dried over anhydrous sodium sulfate. The mixture wasfiltered and solvent removed in vacuo to give 0.374 g of crude product.This was chromatographed on 37 g of silica gel 60 and eluted with ethylacetate to give 0.290 g (88%) of22-([(4-methylphenyl)sulfonyl]oxy)-23,24-bisnorcholane-3α ,7α-diol.

The analytical sample was recrystallized from isopropanol/water. Mp87°-89° C.

[α]²⁵ D=+8.25 (c 0.9933, CHCl₃).

Calcd. for C₂₉ H₄₄ O₅ S: C, 69.01; H, 8.79; S, 6.35. Found: C, 69.21; H,8.88; S, 6.07.

EXAMPLE 922-([(4-methylphenyl)sulfonyl]oxy)-23,24-bisnorcholane-3α,7α-diol3,7-diacetate

A mixture of 0.290 g (0.00049 mole) of22-([(4-methylphenyl)sulfonyl]oxy)-23,24-bisnorcholane-3α,7α-diol, 0.6ml (0.0064 mole) of acetic anhydride, 0.6 ml (0.0074 mole) of drypyridine, 0.003 g (0.000025 mole ) of 4-dimethylaminopyridine, and 10 mlof dry toluene was stirred overnight under an argon atmosphere. Themixture was acidified with 50 ml of 0.5 Normal hydrochloric acid andextracted with 3×20 ml of ethyl acetate. The ethyl acetate extracts werewashed with water until neutral, then dried over anhydrous sodiumsulfate. The mixture was filtered and solvent removed in vacuo to give0.311 g of crude product. This was recrystallized from methanol to giveas a first crop material 0.2577 g (76%) of22-([(4-methylphenyl)sulfonyl]oxy)-23,24-bisnorcholane-3α,7α-diol3,7-diacetate.The mother liquor, after evaporation gave 0.065 g (19%) of product whichby tlc appeared to be greater than 95% pure. The analytical sample wasrecrystallized from methanol. Mp 174°-175° C.

[α]²⁵ D+7.41 (c 0.8768, CHCl₃).

Calcd for C₃₃ H₄₈ O₇ S: C, 67.32; H, 8.22. Found: C, 67.34; H, 8.33.

EXAMPLE 10 3α,7α-(diacetoxy)-24-norcholane-23,23-dicarboxylic aciddiethyl ester

0.264 g (0.0055 mole) of a 50% oil dispersion of sodium hydride waswashed under an argon atmosphere with 3×3 ml of dry pentane. Then, 7.5ml of dry toluene was added. 1.056 g (0.0066 mole) diethylmalonate in 5ml of dry toluene was added dropwise, then heated to reflux and 1.1776 g(0.0020 mole) of22([(4-methylphenyl)sulfonyl]oxy)-23,24-bisnorcholane-3α,7α-diol3,7-diacetatein 10 ml of dry toluene was added dropwise. The mixture was heated toreflux for 20 hours. An additional 0.49 g (0.003 mole) ofdiethylmalonate and 0.050 g (0.001) mole of washed (pentane) sodiumhydride was added and the mixture heated to reflux. After five hours,the cooled mixture was poured into 100 ml of water and extracted with3×60 ml of ethyl acetate. The combined ethyl acetate extracts werewashed with water until neutral and dried over anhydrous sodium sulfate.The mixture was filtered and solvent removed in vacuo leaving a 1.114 gresidue. This was chromatographed on 100 g of silica gel 60 and elutedwith methylene chloride/ethyl acetate (9:1) to give 0.849 g (76%) of3α,7α-(diacetoxy)-24-norcholane-23,23-dicarboxylic acid diethyl ester.The analytical sample was recrystallized from methanol/water. Mp121.5°-123° C.

[α]²⁵ D+23.97 (c 1.0679, CHCl₃).

Calcd for C₃₃ H₅₂ O₈ : C, 68.72; H, 9.09. Found: C, 68.92; H, 9.00.

EXAMPLE 11 3α,7α-Dihydroxy-24-norcholane-23,23-dicarboxylic acid

To a solution of 3 ml of methanol, 5 ml of isopropanol and 0.779 g(0.0139 mole) of potassium hydroxide is added 0.350 g (0.0006 mole) of3α,7α-(diacetoxy)-24-norcholane-23,23-dicarboxylic acid diethyl ester.The mixture was heated to reflux under an argon atmosphere for 4 hrsthen left to stir overnight at room temperature. The alcohol solventswere removed in vacuo and the mixture poured into 50 ml of water andwashed with 3×25 ml of diethyl ether. The aqueous layer was acidifiedand the precipitate collected by suction filtration. 0.252 g (96%) ofthe crude diacid was obtained, Mp 203° C., with decomposition. This wasused directly in the next step without further purification.

EXAMPLE 12 Chenodeoxycholic Acid

A mixture of 0.125 g (0.00028 mole) of3α,7α-dihydroxy-24-norcholane-23,23-dicarboxylic acid, 5 ml of xyleneand 1 ml of dry pyridine was heated to reflux for one hour. The mixturewas cooled, solvents were removed in vacuo and the residue dissolved in25 ml of ethyl acetate. The ethyl acetate solution was washed with 3×10ml of 1 N hydrochloric acid, then with water until neutral. The ethylacetate extract was dried over anhydrous sodium sulfate, filtered andsolvent removed in vacuo to give 0.111 g of crude product. This wasrecrystallized from hexane/ethyl acetate to give (first crop material),0.086 g (76%) of product which had the same spectral properties asauthentic chenodeoxycholic acid and gave no melting point depression. Ananalytical sample was prepared by chromatography on silica gel 60eluting with methylene chloride/methanol/hexane, (2:1:1), thenrecrystallized from ethyl acetate.

We claim:
 1. (5β)-24-Norcholane-3α,7α-diol-23,23-dicarboxylic acid.
 2. Acompound of the formula ##STR12## where Y is C₁₋₃ -alkyl.
 3. Thecompound of claim 2 which is(5β)-24-norcholane-3α,7α-diol-23,23-dicarboxylic acid dimethyl ester. 4.A compound of the formula ##STR13## where Y is C₁₋₃ -alkyl.
 5. Thecompound of claim 4 which is (5β)-24-norcholan-3-one-23,23-dicarboxylicacid dimethyl ester.
 6. A compound of the formula ##STR14## where X ismethyl or p-tolyl.
 7. The compound of claim 6 which is(5β)-7α-hydroxy-22-([(4-methylphenylsulfonyl]-oxy)-23,24-bisnorcholan-3-one.8. (5β)-7α,22-Dihydroxy-23,24-bisnorcholan-3-one. 9.7α,22-Dihydroxy-23,24-bisnorchol-4-en-3-one.
 10. A compound of theformula ##STR15## where X is methyl or p-tolyl.
 11. The compound ofclaim 10 which is22-([(4-methylphenyl)sulfonyl]oxy)-23,24-bisnorcholane-3α,7α-diol.
 12. Acompound of the formula ##STR16## where X is methyl or p-tolyl and R isacyl.
 13. The compound of claim 12 which is22-([(4-methylphenyl)sulfonyl]oxy)-23,24-bisnorcholane-3α,7α-diol3,7-diacetate.
 14. A compound of the formula ##STR17## where R is acyland Y is C₁₋₃ -alkyl.
 15. The compound of claim 14 which is3α,7α-diacetoxy-24-norcholane-23,23-dicarboxylic acid diethyl ester.